Supplementary Figure 1. [ 18 F]FHBG is not retained, phosphorylated, or incorporated into the DNA of...
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Transcript of Supplementary Figure 1. [ 18 F]FHBG is not retained, phosphorylated, or incorporated into the DNA of...
Supplementary Figure 1. [18F]FHBG is not retained, phosphorylated, or incorporated into the DNA of B16 cells. (A-C) For the in vitro studies a panel of cell lines was used: B16 melanoma cells, BaF3 wt and BaF3 with sr39tk over-expressed, primary activated pmel T-cells and activated pmel cells transduced with sr39tk. (D-F) For the in vivo studies B16 and BaF3-sr39tk cells were subcutaneously injected into mice 1 week prior to i.v. injection of [18F]FHBG. After a 1hr uptake, mice were sacrificed and tumors were harvested. The following measurements were made: (A, D) amount of [18F]FHBG retained in cells, (B, E) amount of [18F]FHBG phosphorylated, (C, F) amount of [18F]FHBG incorporated in DNA after 4hr.
B16BaF
3
BaF3-
sr39
tkpm
el
pmel-
sr39
tk0
50
100
150
200
[18F
]FH
BG
(nC
i)
B16BaF
3
BaF3-
sr39
tkpm
el
pmel-
sr39
tk0
50
100
150
200
250
Pho
spho
ryla
ted
[18F
]FH
BG
(nC
i)
B16
BaF3-
WT
BaF3-
sr39
tkpm
el
pmel-
sr39
tk0
510
20
30 0 hr4 hr
[18F
]FH
BG
(nC
i)
B16
BaF3-
sr39
tk0
50
100
150
200
[18F
]FH
BG
(x1
e6 nC
i)re
tain
ed/ c
ell
B16
BaF3-
sr39
tk0
1
2
3
4
5
Pho
spho
ryla
ted
[18F
]FH
BG
(nC
i)
B16
BaF3-
sr39
tk0.0
0.5
1.0
1.5
[18F
]FH
BG
(nC
i)
A. D.
B.
C.
E.
F.
Day 1 Day 3 Day 5 Day 8 Day 10
1.3
0.3
%ID
/g
Day 12 Day 15 Day 17 Day 19 Day 21
1.3
0.3
%ID
/g
Day 1 Day 3 Day 5 Day 8 Day 101.3
0.3
%ID
/g
Day 12 Day 15 Day 17 Day 19 Day 21
1.3
0.3
%ID
/g
Day 1 Day 3 Day 5 Day 8 Day 101.3
0.3
%ID
/g
Day 12 Day 15 Day 17 Day 19 Day 21
1.3
0.3
%ID
/gT T T
T TT T T
TTT
T T T T
T T T T
T T T T
B B B
BBB
B B
B B B
BB B
A B
A A
B B
A A
A
A
A
A
C
C C C
Mouse 1
Mouse 2
Mouse 3
C C C
B
BA
Day 1 Day 3 Day 5 Day 8 Day 101.3
0.3
%ID
/g
Day 12 Day 15 Day 17 Day 19 Day 21
1.3
0.3
%ID
/g
Day 1 Day 3 Day 5 Day 8 Day 10
1.3
0.3
%ID
/g
Day 12 Day 15 Day 17 Day 19 Day 21
1.3
0.3
%ID
/g
Day 12 Day 15 Day 17 Day 19 Day 21
1.3
0.3
%ID
/gB
B
TTT
TT
T TTT
TTTT
A
A A
AA
BB
B
B B
T T T
TTTT A
B
B
Mouse 5
Mouse 6
Mouse 4 Day 1 Day 3 Day 5 Day 8 Day 10
1.3
0.3%
ID/g
B BT T TBBB
CC C
Mouse 7 Day 1 Day 3 Day 5 Day 8 Day 10
1.3
0.3
%ID
/gB B B BT B T
Day 12 Day 15 Day 17 Day 19 Day 21
1.3
0.3
%ID
/g
T T TT B
Supplementary Figure 2. Longitudinal changes in T cell trafficking. Seven mice were imaged with microPET/CT 60 min after i.v. injection with [18F]FHBG. Representative 3-D microPET/CT images of the same mouse on different days are displayed. Red arrows indicate days that mice received gp10025-33/DC vaccine and high dose IL-2. A, axillary LN; B, brachial LN; C, cervical LN; T, tumor
Supplementary Figure 3. Detection of sr39tk+ T-cells over the nonspecific accumulation of [18F]FHBG in B16 tumors. (A) C57BL/6 mice were implanted s.c. with 1 x 105 B16 cells in the right shoulder on day 1. Mice with tumors >1cm in diameter were imaged with [18F]FHBG microPET/CT on day 8. Immediately after imaging, 5x106 sr39tk+ T-cells were injected intra-tumorally. After 30 min, a second dose of [18F]FHBG was injected i.v.. Following a 1hr uptake of the probe mice were again imaged by microPET/CT. (B) Representative 3-D microPET/CT images of three mice with B16 tumors alone (left panels) and the same B16 tumors with sr39tk+ T-cells (right panels). T, tumor. (C) Quantitation of [18F]FHBG in B16 tumors alone and the same B16 tumors with sr39tk+ T-cells.
mou
se #
1
mou
se #
2
mou
se #
30
20
40
60
80
100
B16B16 + pmel-sr39tk
%ID
of [
18F
]FH
BG
A.
B.
1.7
0.1%
ID/g
1.7
0.1
1.7
0.1
%ID
/g%
ID/g
Mouse #1
Mouse #2
Mouse #3
B16 aloneB16 +
sr39tk+ T-cell
B16 melanoma bearing mouse
[18F]FHBG PET
Intratumoral injection of
sr39tk+ T-cells
[18F]FHBG PET
C.
T T
TT
T T